Polyethylenglycol (PEG) is widely used as a water soluble carrier for polymer-drug conjugates. PEG is undoubtedly the most studied and applied synthetic polymer in the biomedical field [Duncan, R. Nature Rev. Drug Discov. 2003, 2, 347-360]. As an uncharged, water-soluble, nontoxic, nonimmunogenic polymer, PEG is an ideal material for biomedical applications. Covalent attachment of PEG to biologically active compounds is often useful as a technique for alteration and control of biodistribution and pharmacokinetics, minimizing toxicity of these compounds [Duncan, R. and Kopecek, J., Adv. Polym. Sci. 57 (1984), 53-101]. PEG possesses several beneficial properties: very low toxicity [Pang, S. N. J., J. Am. Coil. Toxicol, 12 (1993), 429-456], excellent solubility in aqueous solutions [Powell, G. M., Handbook of Water Soluble Gums and Resins, R. L. Davidson (Ed.), Ch. 18 (1980), MGraw-Hill, New York], and extremely low immunogenicity and antigenicity [Dreborg, S, Crit. Rev. Ther. Drug Carrier Syst., 6 (1990), 315-365]. The polymer is known to be non-biodegradable, yet it is readily excretable after administration into living organisms. In vitro study showed that its presence in aqueous solutions has shown no deleterious effect on protein conformation or activities of enzymes. PEG also exhibits excellent pharmacokinetic and biodistribution behavior. [Yamaoka, T., Tabata, Y. and Ikada, Y., J. Pharm. Sci. 83 (1994), 601-606].
In the early developmental stage of PEGylation, the attention has been focused on the amino groups, which are the most represented groups in proteins and are the most suitable conjugation sites. Amino groups are generally exposed in an aqueous environment or other solvent, and can be modified with a wide selection of chemical strategies. Several conjugation strategies are now available, such as alkylation, which maintains the positive charge of the starting amino group because a secondary amine is formed, or acylation, accompanied by loss of charge. [Graham, L. M., Adv. Drug Deliv. Rev. 55 (2003), 1293-1302; Levy, Y. et al., J. Pediatr., (1988) 113, 312-317; Bailon, P. et al., Bioconjug. Chem., 12 (2001), 195-202; Wang, Y. S. et al., Adv. Drug Deliv. Rev. 54 (2002), 547-570; Kinstler, O. B. et al., Pharm. Res., 13 (1996), 996-1002; Wong, S. S., Chemistry of protein conjugation and cross-linking, p. 13 (1991), CRC Press; Caliceti, P. et al., J. Bioact. Comp. Polym. 8 (1993), 41-50]
Esters with PEG have been utilized in chemical modifications of drugs. PEG esters which have an electron withdrawing substituent (alkoxy) in the a-position have proved to be especially effective linking groups in the design of prodrugs since the substituent aids in the rapid hydrolysis of the ester carbonyl bond, thus releasing alcohols in a continuous and effective manner. For instance, highly water soluble PEG-5000 esters of paclitaxel were synthesized and shown to function as prodrugs, i.e., breakdown occurred in a predictable fashion in vitro. [R. B. Greenwald, A. Pendri, D. Bolikal, C. W. Gilbert, Bioorg. Med. Chem. Lett., 4 (1994), 2465-2470]. Studies also showed that amino acid conjugates appeared to be the most useful, reducing toxicity while increasing efficacy for most of the anticancer drugs [A. Pendri, C. D. Conover, R. B. Greenwald, Anti-Cancer Drug Design, 13 (1998), 387-395; R. B. Greenwald, A. Pendri, C. D. Conover, C. Lee, Y. H., Choe, C. Gilbert, A. Martinez, J. Xia, D. Wu, M. Hsue, Bioorg. Med. Chem., 6 (1998), 551-562]
Thiol modification is another potentially useful strategy of PEGylation. For instance, nonessential amino acids in a protein sequence can be replaced by cysteine residues and can be replaced almost anywhere. Such mutant proteins have been generated to PEGylate therapeutically important drugs such as Granulocyte colony-stimulating factor (G-CSF) or human growth hormone (HGH) [Cox, G., Bolder Biotechnology, WO9903887; Berna, M. et al., 32nd Annual meeting & exposition of the controlled release society, 18-22 Jun. (2005), Abstract No. 415, Miami, USA]
Less specific linking strategies relied on the reduction of protein disulphide bridges with the aim of exposing new thiol groups have been used for the PEGylation of antibodies, as amino groups are not suitable as the modification sites because of the marked loss of recognition that occurs during the PEGylation procedure. However, disulphide bridges that link the IgG heavy chains can be cleaved, yielding an active Fab moiety with new exposed thiol groups where the sites of conjugation are also localized far from the antibodies recognition site. [A. P. Chapman et al., Nat. Biotechnol., 17 (1999), pp. 780-783]
In recent years lipid conjugates to PEG have generated great interest, as a result of the discovery that incorporation of PEG-lipids into liposomes yields preparations with superior performance in comparison to conventional liposomes. Such liposomes remain in the blood circulation for extended periods of time and distribute through an organism relatively evenly with most of the dose remaining in the blood compartment and only 10-15% of the dose in liver. This constitutes a significant improvement over conventional liposomes. [Woodle, M. C. and Lasic, D. D., Biochim. Biophys. Acta, 1113 (1992), 171-199]. In these studies, amide-linked mPEG-DSPE (1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine) was prepared by coupling mPEG to the amino group of phosphatidyl ethanolamines [Parr, M. J., Ansell, S. M., Choi, L. S. and Cullis, P. R., Biochim. Biophys. Acta, 1195 (1994), 21-30] and the modified surface amino groups of DSPC-DPPE-cholesterol vesicles were reacted with mPEG-tresylate after liposome formation, instead of using a PEG-lipid conjugate to form liposomes [Senior, J., Delgado, C., Fisher, D., Tilcock, C. and Gregoriadis, Biochim. Biophys. Acta, 1062 (1991), 77-82]. The attractive feature of this approach is in its selective grafting of the polymer on the exterior of the vesicles, thus avoiding the presence of mPEG residues inside the liposomes. A similar study was reported, where grafting mPEG residues onto preformed liposomes, maleimido-PE-containing vesicles were prepared and then reacted with a thiol derivative of PEG [Herron, J. N., Gentry, C A., Davies, S. S., Wei. A. and Lin, J. N., J. Controlled Rel., 28 (1994), 155-166].
Despite all this progress, significant potential for improved liposomal drug delivery remains. So far, few liposomal drugs have been approved for clinical use. Difficulties in obtaining suitable formulations of many drugs remain the challenges. Furthermore, it is desirable to develop new methods and materials to improve manufacturing, cell targeting, and drug release.